Although our understanding of the mechanisms and possible treatment of cancer has increased over recent years, cancer remains a major cause of death throughout the developed world. Non-specific approaches to cancer management, such as surgery, radiotherapy and generalized chemotherapy, have been successful in the management of some circulating and slow-growing solid cancers. However, many types of cancer are generally highly resistant to standard treatments. Accordingly, there is a need for further, and more effective, cancer therapies.
The development of new therapies depends on the identification of suitable targets for drug activity.
The tumour suppressor gene p53 induces cell cycle arrest and promotes apoptosis thereby preventing transformation of cells. Inactivation of the tumour suppressor gene p53 is the most common genetic defect in cancer affecting more than half of all human tumours. The p53 protein is stabilized in response to genotoxic stress, metabolic changes and other potentially dangerous events which can result in transformation of cells.
p63 and p73 are members of the p53 family of transcription factors and have been shown to act in a pathway parallel to that of p53, being up-regulated in response to DNA damage and inducing growth arrest and apoptosis in a p53 independent pathway. Regulation and function of p63 and p73 is reviewed in Melino et al. 2003. They both induce cell cycle arrest and apoptosis and have been recently shown to act as tumour suppressors in vivo (Flores et al., 2005).
The importance of p63 and p73 in tumour suppression is demonstrated by the finding that disruption of p63 and p73 in p53−/− cells increases their transformation capacity. Recently it has been shown that the specific p53 mutations commonly found in cancers such as Li Fraumeni syndrome lead to functional inactivation of p73 and p63 and therefore inactivation of p63 and p73 is relevant to in vivo tumorigenesis (Lang et al, 2004; Olive et al, 2004).
Both p63 and p73, like p53, have a modular structure (FIG. 1A) (Kaghad, 1997). They share a high degree of sequence homology with p53 and can bind to p53-responsive elements activating the transcription of p53 target genes, such as those inducing cell cycle arrest and promoting apoptosis (Catani, 2002; De Laurenzi, 1998; De Laurenzi, 2000).
Unlike p53, however, p73 and p63 are expressed as different isoforms (Kaghad, 1997; Ueda, 1999) some of which lack the transactivation domain and are believed to act as dominant negative proteins (Melino, G et al.). Most of the variation generated by alternative splicing occurs at the 3′ end, in a part of the sequence that does not have a counterpart in p53. The existence of these variant isoforms has made it difficult to determine the importance of p63 and p73 in tumour suppression. However, recent work using p63/p73 mutant mice has clearly demonstrated that both these proteins have tumour suppressor functions independent of p53. In addition, p63 and p73 mutant mice are predisposed to aggressive epithelial tumours common in humans (e.g. lung and mammary adenocarcinomas), unlike p53 mutant mice, which primarily develop thymic lymphomas and sarcomas (Flores et al, 2005).
At least six different p73 proteins (a to TI) are generated (De Laurenzi, 1999; De Laurenzi, 1998; Ueda, 1999) while at least three different p63 proteins are generated as alternatively spliced C-terminal isomeric forms. In addition, both p63 and p73 genes exploit an alternative promoter and an extra exon (exon 3′) to generate N-terminally truncated isoforms (ΔNp63 and ΔNp73). These variants lack the transactivation domain and act as “dominant negatives”, blocking the function of either p53, p63 or p73 full-length proteins (Grob, 2001; Sayan et al., 2004; Yang, 2000). The relative levels of TA and ΔN isoforms determine cell fate, resulting in either growth arrest and death or uncontrolled proliferation.
TAp73 steady state protein levels are up-regulated in response to DNA damage in a fashion distinct from p53 (Agami, 1999; Gong, 1999; Yuan, 1999) while ΔNp73 is rapidly degraded (Maisse et al., 2004). ΔNp63 expression is transcriptionally reduced by p53 suggesting that it does not inhibit the tumour suppression activity of p53 and TAp73 in the same way. These observations suggest an important differential role for these isomers in carcinogenesis (Melino, 2002; Melino et al., 2003; Stiewe, 2002; Zaika, 2002).
The role of p63 and p73 in cell cycle and apoptosis suggests that their modification can contribute to enhanced cell death in tumours. In addition, several mutations in p63 are associated with genetic epidermal syndromes while p73 overexpression is sufficient to trigger neuronal differentiation. Accordingly, modification of p63 or p73 stability may be a therapeutic strategy in the treatment of cancer and/or developmental disorders.
While ubiquitination and proteasomal-dependent degradation of p53 is regulated by its transcriptional target MDM2, the regulation of p73 and p63 protein degradation is controlled by distinct E3 ligases. To date very little is known of the molecular mechanisms underlying the regulation of p63 and p73 protein steady state levels and their modulation as a possible therapeutic strategy has not been fully explored. While some approaches have sought to use proteosome inhibitors to inhibit the degradation of p53-family proteins and thus induce apoptosis, in clinical trials these inhibitors have been found to have very little specificity and lead to up-regulation of a large number of proteins. Moreover, while a number of cancer therapeutic strategies have targeted p53, more than 50% of tumours are p53 deficient. Accordingly, there is a need for therapeutics that can target p53 independent pathways.